During the last grant cycle, we have characterized a kinetic intermediate in the binding of antigenic peptides to MHC class II molecules. These intermediate complexes are characterized by low affinity and fast association and disassociation rates; they represent peptide-receptive forms, and require a conformational change to occur before converting to stable complexes. The intermediate complexes can be mimicked by variant of flu HA 306-318 peptide (changes to P1-binding residues) and by an HLA-DR1 molecule mutated in the P1 pocket. During the next cycle, we propose to study the biology of these short-lived intermediate complexes, addressing three aims: 1. Characterization of in vitro T cell responses to short-lived complexes, expected to be anergy induction. 2. Identify APCs responsible for anergy induction 3a. Compare peptide repertoires of DO-/-, DM-/- and WT thymic stromal cells 3b. Generate mice expressing a DR1 mutant (DRbG86Y) that participates in short-lived complexes and study thymocyte selection.